1021-4437/02/4903- $27.00 © 2002
Russian Journal of Plant Physiology, Vol. 49, No. 3, 2002, pp. 320–325. Translated from Fiziologiya Rastenii, Vol. 49, No. 3, 2002, pp. 359–364.
Original Russian Text Copyright © 2002 by Tikhaya, Fedorovskaya.
Cyclic AMP is a second messenger with a broad
spectrum of activity in bacterial, fungal, and animal
cells . In particular, cAMP plays an important role in
the intercellular communication in mixomycetes  and
in the Ca
-dependent processes in animal cells [3, 4].
Concerning plant cells, there are some experimental
data indicating that cAMP treatment can induce a cas-
cade of metabolic changes. The most bright examples
of exogenous cAMP effects on various physiological
processes are stimulation of lettuce seed germination,
elongation of lettuce hypocotyls and oat coleoptiles,
pollen tubes, etc. . On
the other hand, cAMP inhibited the formation of gall
tumors on bean leaves and suppressed the growth of
appressoria of the rice fungal pathogen . In addition,
recent studies performed with cultured cells supported
convincingly an important role of cAMP in the control
inﬂux into the cell .
Some other data indicate the involvement of cAMP
in the control of biochemical reactions in the plant cell
cytosol. Thus, cAMP was capable of changing enzy-
matic activities: it activated acid phosphatase, nitrate
reductase, and polyphenol oxidase. In other cases, it
inhibited acid phosphatase, 5'-nucleotidase, and 5',3'-
ribonucleotide phosphohydrolase [5, 7].
The effects of cAMP on apoplastic enzymes are not
studied, although they can be expected. Such enzymes
were detected on the surface of root cells [8–10]. In par-
ticular, we demonstrated the presence of AP and nucle-
otidases in barley root cells . One nucleotidase
demonstrated a high afﬁnity for ATP and Ca
another one, for AMP and Mg
. The aforementioned
enzymes were bound to the outside of the plasma-
lemma and were also able to be in the membrane-free
The objective of this work was to study the
effects of cAMP on activities of membrane-bound and
soluble acid phosphohydrolases and their substrate
speciﬁcity in barley root cells.
MATERIALS AND METHODS
Roots of ﬁve-day-old etiolated bar-
L.) plants grown on 0.25 mM
under continuous aeration were used in the
Isolation of puriﬁed microsomal fraction.
were homogenized in four volumes of medium contain-
ing 25 mM Tris–HCl, pH 7.5, 250 mM sucrose, and
1 mM EDTA. The homogenate was centrifuged succes-
sively at 8000
for 5 min, 13000
for 15 min, and
for 30 min. The pellet (crude microsomal frac-
tion) was resuspended in 18% sucrose, layered onto a
two-step sucrose gradient consisting of 35 and 45%
sucrose and centrifuged at 95000
for 2 h. The puriﬁed
microsomal fraction was collected from the interface
between these sucrose layers using a needle with the
end bent at a right angle.
Preparation of KCl extracts.
The puriﬁed microso-
mal fraction was extracted with 0.5 M KCl in the cold
for 45 min under continuous stirring. The extract was
centrifuged at 150000
for 1 h, and then supernatant
(KCl extract) was used .
The Effect of cAMP on Phosphohydrolase Activity
of the Barley Root Cells
N. I. Tikhaya and M. D. Fedorovskaya
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;
fax: 7 (095) 977-8018; e-mail: firstname.lastname@example.org
Received April 25, 2001
—We studied the effects of cAMP on phosphohydrolase activity in the microsomal fraction and its
0.5 M KCl extract. In both preparations, cAMP inhibited the activity of acid phosphatases. In the microsomal
fraction, a 50% inhibition of acid cation-dependent (Mg
) phosphatase was induced at pH 6.0 by 10
cAMP; whereas, for cation-independent phosphatase, this effect was exerted only by 50
M cAMP. In the KCl
extract, the sensitivity of both cation-dependent and cation-independent acid phosphatases to cAMP was higher
than in the microsomal fraction. Cyclic AMP did not reduce the activity of nucleosidases. Selectivity in the
cAMP action on acid phosphatases is assumed to be one of the mechanisms controlling the biochemical activity
outside the barley root cells.
Key words: Hordeum vulgare - root cells - phosphohydrolase - cAMP
: AP—acid phosphatase; GP—glycerophosphate;